The invention relates to a method for measuring the aggregation or agglutination of platelets, where a reaction mixture is mixed in a first reaction phase, and is mixed less vigorously or not at all in a second reaction phase following the first, and the measurement is preferably carried out in the second reaction phase.
Blood consists essentially of the blood plasma, a solution of low molecular weight substances and proteins, and cellular constituents, of which the erythrocytes (red blood corpuscles), leukocytes (white blood corpuscles) and platelets (thrombocytes) account for the largest proportion.
Platelets have the main function in vivo of responding rapidly to an injury and initiating the first steps in wound closure. They are activated by various factors and become during the course of activation xe2x80x9cstickyxe2x80x9d, as it has appropriately been called. They then adhere both to injured surfaces and to one another. The adhesion to one another is called aggregation when it takes place actively, and agglutination when it proceeds passively, that is to say without physiological activity.
Hereinafter and, in particular, also in the claims, on use of the term xe2x80x9caggregationxe2x80x9d both aggregation and agglutination of platelets is meant.
The ability to aggregate is thus important for wound closure. It can be enhanced or diminished by a large number of factors. These include, for example, diseases, operations, intake of medicines and the number of platelets. An enhancement of the tendency to aggregate may lead to thromboses and a diminishment in the tendency to aggregate may lead to hemorrhages.
In order to investigate the aggregation properties of platelets, O""Brian (J. Clin. Path., 1962, 15, 452-455) and Born (J. Physiol., 1963, 168, 178-195) developed a method for measuring platelet aggregation which has become widely used in clinical diagnosis in the subsequent decades (Bick R. L., Clinics in Laboratory Medicine, Thrombosis and Hemostasis for the clinical laboratory: Part II, Volume 15, 1, 1-38). For the method, initially platelet-rich plasma is produced. An activator is usually added to the mixture, and the extinction is followed while stirring continuously. The formation of aggregates can be followed quantitatively through the decrease in the extinction. Special instruments, called aggregometers, are used for the measurement, because conventional photometers do not have a controlled stirring mechanism and appropriate modes of evaluation.
The advantage of the method is that the aggregation is measured quantitatively and kinetically, and automation is possible relatively easily. A disadvantage is that specialized instruments with a stirring mechanism during the measurement are required. These instruments, the aggregometers, are costly and acquisition is not worthwhile for every laboratory. Since the number of measurements is often limited, no completely automated instruments have become widely used, and operation is correspondingly inconvenient. On the other hand, a large number of automatic analyzers with a very high degree of automation exist and could in principle, because of the photometric measurement unit, measure the decrease in extinction, but do not have the necessary continuous stirring option.
According to the current state of the art, continuous stirring is necessary for measuring platelet aggregation. As the study by Born (1963) has already shown, the rate of aggregation depends greatly on the stirring rate. More recent review articles on the method assume that the need for stirring is self-evident (Yardumian D. A., J. Clin. Pathol, 39, 1986, 701-712. Bick R. L., Thrombosis and Hemostasis for the Clinical Laboratory: Part II, 15, 1995, 1-38).
The stirring brings about, presumably through an increase in the frequency of collisions, the formation of larger aggregates, called macroaggregates. Macroaggregates are aggregates of platelets which lead to a fall in the extinction in the aggregometer. They are distinctly larger than microaggregates, whose formation is indicated either by no change in the extinction or even by an increase in the extinction (Kitek A. and Bredding K., 1980, Thromb and Haemost, 44, 1980, 3, 154-158). Microaggregates consisting of 2-3 platelets arise even without stirring (Longmire K. and Frojmovic M., Biophys. J, 1990, 58(2): 299-307). However, these microaggregates cannot be seen in conventional aggregometers (Toghi et al., Thromb Haemost, 1996, 75(5): 38-43).
The formation of macroaggregates also proceeds very similarly in the von Willebrand ristocetin cofactor test as in the aggregation of platelets in fresh platelet-rich plasma. In the ristocetin cofactor test, fixed platelets are induced to agglutinate by addition of plasma which contains von Willebrand protein, and ristocetin (Weiss H. J. et al., J. Clin. Invest., 1973, 52, 2708-2716). The turbidimetric measurement with stirring in the ristocetin cofactor test also shows mainly the formation of macroaggregates (Kitek A. and Breddin K., 1980, Thromb and Haemost, 44, 1980, 3, 154-158. Toghi et al., Thromb Haemost, 1996, 75(5): 38-43).
The present invention was thus based on the object of finding a method of measurement with which it is possible to measure platelet aggregation even without continuous stirring.
This object is achieved by the method of the invention for measuring the aggregation of blood platelets (platelets), where a reaction mixture of platelets, preferably physiologically active platelets or fixed platelets, is mixed with other test mixture components in a first reaction phase, and is mixed less vigorously or not at all in a second reaction phase following the first. The platelet aggregation or agglutination is preferably measured in the second reaction phase. Further preferred embodiments of the invention are described in detail in claims 2-17.
It has been found, surprisingly, that the formation of macroaggregates does not necessarily require continuous stirring; on the contrary a short stirring time after addition of the platelet activator is sufficient to induce formation of macroaggregates. The formation of the macroaggregates takes place after the induction phase solely by the action of Brownian diffusion.